Yeast transformant producing recombinant human parathyroid hormone and method for producing the hormone

ABSTRACT

Novel  Saccharomyces Cerevisiae  mutant strains are provided for producing human parathyroid hormone. The novel strains acre genetically disrupted in at least one of the genes encoding the yapsin family of proteases Yapsin 1, Yapsin 2 and Yapsin 3 and harbor a human parathyroid hormone gene in their genomes. Culturing the novel strains results in the secretion of intact hPTH into culture media at high yield.

RELATED APPLICATIONS

This is the U.S. National Phase under 35 U.S.C. §371 of InternationalApplication PCT/KR01/01447, filed Aug. 27, 2001, designating the U.S.and published in English, which claims priority to a Korean patentapplication No. 2000-0051267, filed Aug. 31, 2000, both of which areincorporated herein in their entireties.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the production of human parathyroidhormone (hereinafter referred to as “hPTH”). More particularly, thepresent invention relates to the use of Saccharomyces cerevisiae mutantstrains which are genetically disrupted in at least one of the yapsinfamily of aspartic proteases YPS1, YPS2 and YPS3 and transformed with anexpression vector anchoring a hPTH gene, in producing the hormone.

2. Description of the Prior Art

hPTH is a peptide consisting of 84 amino acid residues, produced by theparathyroid gland. hPTH maintains calcium homeostasis in the kidneys andbones, having the physiological function of promoting calcium metabolismand osteogenesis. In the U.S.A. and the Europe, estrogen or calcitoninhave been predominantly used as therapeutics for osteoporosis, but arefound to be obstructive of bone absorption. Accordingly, leadingpharmaceutical companies of the world are intensively and extensivelystudying hPTH for the development of therapeutics for osteoporosis bytaking advantage of the ability of hPTH to promote osteogenesis.Particularly, more earnest attention is paid to the prophylaxis andtreatment of osteoporosis as modern society becomes an aging society.For above reasons, active research is now being directed to thedevelopment of biotechnology methods for the mass production of hPTH,which is expected to be a promising therapeutic substitutive forconventional therapeutics for osteoporosis.

For example, various attempts have been made to develop methods formass-producing recombinant hPTH by using E. coli as a host cell becausethe prokaryotic bacteria shows relatively high expression efficiency.However, there is a great difficulty in refolding and purifying therecombinant proteins produced from E. coli. In contrast, yeast, a singlecell eukaryote, has the advantage of expressing and secreting properlyfolded- and thus active-proteins because it is very similar to higherspecies in the gene transcription and translation systems, and itsprotein-secretion system. Additionally, yeast is advantageous as a hostfor producing proteins of interest in that yeast secretes fewextracellular proteins making it easy to recover and purify exogenousproteins. Further, the yeast Saccharomyces cerevisiae is a GRAS(generally recognized as safe) microorganism that is not pathogenic tothe body and does not produce endotoxins. With the anticipation of beingvery useful as a producer of medicinal recombinant hPTH, Saccharomycescerevisiae has been studied in developing hPTH expression systems. Inspite of its various advantage as an expression host for the productionof recombinant hPTH, Saccharomyces cerevisiae is not industriallyutilized as such a host because the extracellularly secreted recombinanthPTH is degraded by endogenous proteolytic enzymes of the yeast's own,so that only a small amount of the intact molecule of hPTH can berecovered (Gabrielsen et al., Gene 90, 255(1990)).

In the last decade, extensive studies have been made for solving theproteolysis problem of recombinant hPTH. For instance, based on thefinding that hPTH is cleaved between Arg-25 and Lys-26, which isidentical to the recognition site of KEX2, a protease present in yeastGolgi bodies, a substitution mutant of hPTH, which has glutamine atposition 26 of its amino acid sequence instead of lysine, was made withthe aim of preventing the proteolysis by KEX2, which is a putativeprotease to cleave hPTH (Reppe et al., J. Biol. Chem. 266, 14198(1991)). Using gene recombination technology, an hPTH-related proteinwas directly ligated to the 3′-end of the yeast ubiquitin gene toproduce a non-cleavable hPTH-related protein (Rian et al., Eur. J.Biochem., 213, 641 (1993)). However, if protein mutants are medicinallyused, there are required stringent tests to obtain permission for theirmedicinal use, because they are generally recognized as new medicines.When using gene fusion technology, it is necessary to remove the fusionsite by expected digestion because proteins of interest may be producedat relatively yields owing to the presence of the fusion site.

As a result of the research for the prevention of hPTH degradationwithout resorting to hPTH protein mutants or fusions, the presentinventors developed a method in which hPTH cleavage can be prevented toa significant extent simply by adding L-arginine at high concentrationsto the culture media (Chung and Park, Biotechnol. Bioeng. 57, 245(1998)). The fact that hPTH cleavage is prevented to a significantextent by the presence of L-arginine in the culture media indicates thathPTH cleavage is performed mainly by extracellular proteases rather thanby the intracellular protease Kex2p. From this finding, the presentinventors inferred that Yap3p (yeast aspartic protease 3), which, likeKEX2, is able to cleave the C-terminal or middle sites of basic singleor couple amino acids, and binds to the cytoplasmic membrane of yeast,is practically responsible for the cleavage of the hPTH secreted intoyeast culture media. On the basis of this inference, the presentinventors made a YAP3 gene-disrupted yeast strain (yap3Δ), andconstructed an hPTH production system by use of the yeast mutant. Uponculturing in flasks, the hPTH production system was found to prevent thecleavage of hPTH at an efficiency of as high as 80%, thus producing theintact molecule of hPTH at high yield (Kang et al., Appl. Microbiol.Biotechnol., 50, 187 (1998); Korean Pat. No. 0246932, yielded Dec. 8,1999). Though exhibiting far higher hPTH productivity than the wild typestrain, the yap3Δ mutant was observed to allow hPTH to be cleaved to asignificant extent in the late stage of the high-concentration culturing(Song and Chung, Process Biochem 35, 503 (2000)), which indicates thathPTH is cleaved not by Yap3p, but by other proteases in the late culturestage. Saccharomyces cerevisiae is reported to have the asparticprotease MKc7p, which is very similar in structure and function to Yap3p(Komano and Fuller, Proc Natl Acad Sci, USA, 92, 10752 (1995)). Thepresent inventors created a yeast mutant in which both of the genes aredisrupted (yap3Δ/mkc7Δ) for use in the observation of the influence ofthe enzymes on the hPTH cleavage at the terminal culture stage. However,no significant differences were found between the mutants yap3Δ andyap3Δ/mkc7Δ (Choi, et al., J. Microbiol Biotechnol 9, 679 (1999)). Theseresults demonstrated that the MKc7p protease, through having highhomology with Yap3p (53% homology) and being involved in the processingof pro-α-mating factor in the absence of Kex2p, is not greatlyresponsible for hPTH cleavage.

Through the analysis of the recently disclosed genome information aboutSaccharomyces cerevisiae, a search was made for genes homologous to YAP3(recently renamed YPS1), resulting in the finding that three novel genescoding for unknown aspartic proteases, in addition to PEP4 and BAR1, arepresent (Olsen et al., Biochem. J. 339, 407 (1999)). Assumed to encodenew members of the yapsin family of aspartic proteases, like YPS1 andYPS2, the three novel genes were named YPS3, YPS6 and YPS7,respectively. YPS3 was found to have 50% homology with both of YPS1 andYPS2, while 35% and 25% homology was found between YPS6 and BAR1 andbetween YPS7 and PEP4, respectively. From the homology between YPS3 andYPS1, the present inventors drew the deduction that the hPTH cleavageoccurring at the terminal culture stage of the yps1Δ (previous yap3Δ)strain might be performed by yapsin 3.

SUMMARY OF THE INVENTION

With the background in mind, the present invention has an object ofproviding a protein expression system, which can produce hPTH at highyield.

It is another object of the present invention to provide a method forproducing hPTH at high yield.

Knowledge of the posttranslational modification of hPTH allowsmodification and adaptation leading to the present invention.

As a result of intensive and thorough research on the biologicalproduction of hPTH, the present inventors found that the disruption ofyapsin genes results in a surprising decrease in the endogenousdegradation of recombinant hPTH in Saccharomyces cerevisiae.

In accordance with an aspect of the present invention, there is provideda Saccharomyces cerevisiae mutant strain in which both of YPS1 and YPS3genes or all of YPS1, YPS2 and YPS3 genes are disrupted.

In accordance with another aspect of the present invention, there isprovided a method for producing hPTH by using the Saccharomycescerevisiae mutant strain as a producer.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects, features and other advantages of thepresent invention will be more clearly understood from the followingdetailed description taken in conjunction with the accompanyingdrawings, in which:

FIG. 1 is a diagram showing a process of constructing a cassette fordisrupting the YPS3 gene by use of a pop-out URA3 selection marker;

FIG. 2 is a diagram showing a process of disrupting the YPS3 gene ofSaccharomyces cerevisiae and recovering the URA3 selection marker;

FIG. 3 shows SDS-PAGE results of hPTH molecules obtained from thecultures of Saccharomyces cerevisiae 2805 (lane 1), Saccharomycescerevisiae 2805/pG10-hPTH1 (lanes 2 and 3), Saccharomyces cerevisiaeSY28Y3/pG10-hPTH1 (lanes 4 and 5), and Saccharomyces cerevisiaeSY28Y4/pG10-hPTH1 (lanes 6 and 7), along with 1 μg of native hPTH (C)and a prestained protein molecular weight marker (M) grown for 24 hours(a) and 48 hours (b), wherein band i stands for intact hPTH (1-84 a.a.),band d1 for a truncated hPTH (27-84 a.a.), and band d2 for hPTH (1-80a.a.);

FIG. 4 shows SDS-PAGE results of hPTH molecules obtained from thecultures of Saccharomyces cerevisiae L3262 (lane 1), Saccharomycescerevisiae L3262/pG10-hPTH1 (lanes 2 and 3), Saccharomyces cerevisiaeSLH15/pG10-hPTH1 (lanes 4 and 5), Saccharomyces cerevisiaeSLH16/pG10-hPTH1 (lanes 6 and 7), Saccharomyces cerevisiaeSLH17/pG10-hPTH1 (lanes 9 and 9), and Saccharomyces cerevisiaeSLH18/pG10-hPTH1 (lanes 10 and 11), along with 1 μg of native hPTH (C)and a prestained protein molecular weight marker (M) grown for 24 hours(a) and 48 hours (b), wherein band i stands for intact hPTH (1-84 a.a.),band d1 for a truncated hPTH (27-84 a.a.), and band d2 for hPTH (1-80a.a.); and

FIG. 5 shows SDS-PAGE results of hPTH molecules obtained from thecultures of Saccharomyces cerevisiae L3262 (lane 1), YPS1/YPS3-doubledisruptants of Saccharomyces cerevisiae L3262 (a): Saccharomycescerevisiae L3262/pG10-hPTH1 (lanes 2 to 7) and Saccharomyces cerevisiaeSLH11/pG10-hPTH1 (lanes 8 to 13), YPS1/YPS2/YPS3-triple disruptants (b)of Saccharomyces cerevisiae L3262: Saccharomyces cerevisiaeSLH16/pG10-hPTH1 (lanes 2 to 7) and Saccharomyces cerevisiaeSLH18/pG10-hPTH1 (lanes 8 to 13), along with 1 μg of native hPTH (C) anda prestained protein molecular weight marker (M) grown for 72 hours withcontinual supply of galactose, wherein band i stands for intact hPTH(1-84 a.a.), band d1 for a truncated hPTH (27-84 a.a.), and band d2 forhPTH (1-80 a.a.).

DETAILED DESCRIPTION OF THE INVENTION

The present invention pertains to a method for producing recombinanthPTH from a yeast host which is unable to produce at least one of theyapsin family of proteases YPS1, YPS2 and YPS3.

Showing exoprotease activity to cleave residues from the C- or N-terminiof hPTH, the protease Yapsin 1 (hereinafter referred to as “YPS1”),Yapsin 2 (hereinafter referred to as “YPS2) or Yapsin 3 (hereinafterreferred to as “YPS3”) makes it difficult to produce the intact moleculeof hPTH in yeast host. Accordingly, the present invention comprises amethod for producing the intact molecule of hPTH at a high yield byusing as a host cell a yeast mutant which is unable to express at leastone of the three proteases YPS1, YPS2 and YPS3.

It is believed that YPS1 and YPS2 exert their enzymatic activity mostlyat the early culture stage of transformants for producing hPTH, whileYPS3 cleaves hPTH mainly at the late culture stage. In the case of theyps1Δ strain in which the YPS1 gene is disabled, hPTH can be obtained ata high yield in the early culture stage because of the lacking of YPS1,but at a poor yield in the late culture stage owing to the inevitableproteolytic activity of YPS3.

Accordingly, in order to improve the production yield of hPTH throughoutthe culturing of a yeast host, a mutant defective preferably in bothYPS1 and YPS3 (yps1Δ/yps3Δ), and more preferably in all of YPS1, YPS2and YPS3 (yps1Δ/yps2Δ/yps3Δ) is used as the host.

Deletion of the proteases can be carried out by disrupting at least onegene selected from the group consisting of YPS1, YPS2 and YPS3 by use ofan enzyme selection marker.

The yeast selection marker is not particularly limited, but ispreferably a one that can pop-out. In a preferred embodiment of thepresent invention, a URA3 selection marker is provided in a form of acassette. The pop-out cassette containing a yeast selection marker hasgene fragments coding for N- and C-terminal sites of YPS1, YPS2 and YPS3at its both ends such that the target genes contained in the genome ofyeast, i.e., yps1, yps2 and yps3, can be disrupted by a homologyrecombination method. Any selection marker may be used if it is able toselect the yeast which harbors the cassette in its genome. Herein, themutant yps3Δ in which the yps3 gene is disrupted by a URA3 selectionmarker is denoted by yps3::URA3.

In accordance with the present invention, an hPTH gene is carried intoyeast by an expression vector. Accordingly, the present inventionpertains to a recombinant expression vector to which an hPTH gene isinserted. The expression vector useful in the present invention hasmeans for expressing a gene in yeast and means for controlling theexpression. Vector selection and recombinant vector construction areobvious to those who are skilled in the art and details thereof will bedescribed in the following examples.

In another aspect, the present invention pertains to a transformantprepared from the yps1Δ/yps3Δ or yps1Δ/yps2Δ/yps3Δ yeast mutant with therecombinant expression vector.

In a preferred embodiment of the present invention, the recombinantvector pG10-hPTH 1 is transformed into Suecharomyces cerevisiae mutants(ypsIΔ/yps3Δ and yps1Δ/yps2Δ/yps3Δ) to create transformantsSLH16/pG10-hPTH and SLH18/pG10-hPTH, which were deposited in the KoreanCollection for Type Culture of Korea Research Institute of Bioscienceand Biotechnology (KRIBB) (#52, Oun-dong, Yusong-ku, Taeion 305-333,Republic of Korea) with accession Nos. KCTC 0815BP and KCTC 0816BP,respectively, on Jul. 6, 2000. This deposit was made under theprovisions of the Budapest Treaty on the International Recognition ofthe Deposit of Microorganisms for the Purposes of Patent Procedure andthe Regulations thereunder (Budapest Treaty). This assures maintenanceof a viable culture of the deposit for 30 years from date of deposit.The deposit will be made available by KRIBB under the terms of theBudapest Treaty, and subject to an agreement between Applicant and KRIBBwhich assures permanent and unrestricted availability of the progeny ofthe culture of the deposit to the public upon issuance of the pertinentU.S. patent or upon laying open to the public of any U.S. or foreignpatent application, whichever comes first, and assures availability ofthe progeny to one determined by the U.S. Commissioner of Patents andTrademarks to be entitled thereto according to 35 USC § 122 and theCommissioner's rules pursuant thereto (including 37 CFR § 1.14).Availability of the deposited strain is not to be construed as a licenseto practice the invention in contravention of the rights granted underthe authority of any government in accordance with its patent laws.

A better understanding of the present invention may be obtained in lightof the following examples which are set forth to illustrate, but are notto be construed to limit the present invention.

Experimental Strain and Plasmid

For the expression of hPTH in Saccharomyces cerevisiae, there wasemployed the plasmid pG10-hPTH1, which contains an hPTH expressioncassette composed of GAL10 promoter::ppL::hPTHdb?::GAL7 terminator(Chung and Park, Biotechnol. Bioeng. 57, 245 (1998)). A pop-out URA3selection marker (URA3::tc5) cassette was prepared from a 1.8 kb BamHIfragment derived from pTcUR3 (Kang et al., Appl. Microbiol. Biotechnol.,53, 575-582 (2000)). Saccharomyces cerevisiae 2805 and Saccharomycescerevisiae L3262a were used as parental cells for preparing YPS3-deletedmutants (Kang et al., J. Microbiol. Biotechnol., 8, 42-48 (1998)). Also,the cells used in the present invention were the yeast strains SLH11(Kang et al., Appl. Micribiol. Biotechnol., 59, 187 (1998)), SLH12 andSLH14 (Choi et al., J. Biosci. Bioengin., 89, 77 (2000)), which aredefective in the YPS1 gene (previously YAP3), the YPS2 gene (previouslyMKC7), or both of them. Genetic characteristics of the yeast strains aresummarized in Table 1, below.

TABLE 1 Saccharomyces cerevisiae Strains Used in the Invention StrainDescription/Genotype 2805 a parental strain (MATα pep4::His3 prb-Δ1.6Rcan1 his3-20 ura3-52) S28Y3 a yps1-disrupatant of 2805 (MATα pep4::His3prb-Δ1.6R can1 his3-20 ura3-52 yps1::tc5) L3262 a parental strain (MATaura3-52 leu2-3, 112 his4-34) SLH11 a yps1-disruptant of L3262 (MATaura3-52 leu2-3, 112 his4-34 yps1::LEU2) SLH12 a yps2-disruptant of L3262(MATa ura3-52 leu2-3, 112 his4-34 yps2::LEU2) SLH14 a yps3-disruptant ofL3262 (MATa ura3-52 leu2-3, 112 his4-34 yps1::HIS4 yps2::LEU2)

Medium Composition and Culture Condition

When transforming yeast strains with a URA3 cassette for disrupting theYPS3 gene or with the expression vector pG10-hPTH1, the synthetic mediumSC-URA which was deficient in uracil only was used. To recover the URAselection marker from the obtained yps3:: URA3 mutant, a 5-FOA(5-fluorotate) medium was employed (Adams et al., Methods in yeastgenetics. Cold Spring Harbor Laboratory Press, 1997). For use in theinduction of hPTH expression under the control of the GAL 10 promoter,cultures were grown for 48 hours on YPDG media (yeast extract 1%, Bactopeptone 2%, glucose 1%, galactose 1%).

EXAMPLE 1 Establishment of YPS3 Gene-Disrupted Yeast Mutant

The construction of a recombinant vector containing a URA3 cassette foruse in disrupting the YPS3 gene is illustrated in FIG. 1.

N- and C-terminal fragments of YPS3 gene necessary for the homologyrecombination leading to the disruption of the YPS3 gene were producedby PCR (polymerase chain reaction) using two pairs of primers, whichwere synthesized on the basis of the YPS3 base sequence of Saccharomycescerevisiae, registered in GenBank. For the convenience of genemanipulation later, a set of primers (5′-GACGAATTCCAGAAACGTCTGAGTGGAG-3′(SEQ ID NO: 1) and 5′-GCAGGATCCGTACTCTACCGAATGCCG-3′ (SEQ ID NO: 2)) foramplifying the N-terminal fragment were designed to have recognitionsites of EcoRI and BamHI at their respective 5′-terminal sites(underlined parts) while restriction sites of BamHI and XbaI wereintroduced into 5 ′-terminal sites of a set of primers(5′-CGCGGATCCCTATGCAGACCAGTGTGG-3′ (SEQ ID NO: 3) and5′-CGCTCTAGACTGCATGCAAGGTCTGAC-3′ (SEQ ID NO: 4)) for the amplificationof the C-terminal fragment (underlined parts). The PCR was carried outin a thermal cycler, such as that manufactured by Perkin Elmer,identified as “GeneAmp PCR 24001”, with 25 thermal cycles, eachconsisting of 95° C./30 sec for denaturing, 55° C./30 sec for annealing,and 72° C./30 sec for extending, so as to produce a 800 bp and a 700bp-DNA fragment encoding an N-terminal and a C-terminal region of theYPS3 gene region, respectively, from the genomic DNA of Saccharomycescerevisiae. The PCR products, that is, the YPS3 N-terminal andC-terminal fragments, were double digested with restriction enzymesEcoRI/BamHI and BamHI/XbaI, respectively, followed by ligating the tworestriction enzyme digests together into the pBluescript II KS(+) vector(Stratagen) which was previously treated with EcoRI/XbaI. To the BamHIrecognition site located between the two terminal fragments of theresulting recombinant vector pB-YPS3NC, a URA3 popout selection markerwas introduced, so as to construct the 15 pB-yps3:: URA3: tc5 vector foruse in disrupting the YPS3 gene.

Next, the YPS3 gene of Saccharomyces cerevisiae was disrupted by use ofthe URA3 cassettes, followed by recovering the URA3 selection markertherefrom, as illustrated in FIG. 2.

In detail, after the DNA fragment obtained by the enzyme restrictiontreatment of the vector pB-yps3:: URA3: tc5 with EcoRI/XbaI wastransfected into Saccharomyces cerevisiae strains 2805, L3262a, SLH11(yps1Δ), SLH12 (yps2Δ), and SLH14 (ypsΔ/yps2Δ), primary selection forUra⁺ transformants was made on SC-URA selection media. PCR was conductedto identify whether the Ura⁺ transformants were disrupted in the YPS3gene, after which the yps3:: URA3: tc5 transformants thus selected werespread onto 5-FOA plates to select the yps3:: tc clones resulting fromthe pop-out of the URA 3 gene through homology recombination.

The disruption of the YPS3 gene in the finally obtained yps3Δ mutantswas identified again by PCR to obtain yps3-disruptants, S28Y4, SLH15,SLH16, SLH17 and SLH18, which were optionally defective in other yapsinprotease genes. These mutant stains are summarized in Table 2, below.

TABLE 2 Yps3-Deleted Yeast Strains Strain Description/Genotype S28Y4 ayps3-disruptant of 2805 (MATα pep4::His3 prb-Δ1.6R can1 his3-20 ura3-52yps3::tc5) SLH15 a yps3-disruptant of L3262 (MATa ura3-52 leu2-3, 112his4-34 yps3::tc5) SLH16 a yps1/yps3-double disruptant of L3262 (MATaura3-52 leu2-3, 112 his4-34 yps1::LEU2 yps3::tc5) SLH17 ayps2/yps3-double disruptant of L3262 (MATa ura3-52 leu2-3, 112 his4-34yps2::HIS4 yps3::tc5) SLH18 a yps1/yps2/yps3-triple disruptant of L3262(MATa ura3-52 leu2-3, 112 his4-34 yps1::LEU2 yps2::HIS4 yps3::tc5)

EXAMPLE 2 Establishment of Recombinant Yeast Strain Expressive of hPTHand Analysis of hPTH Expression

Saccharomyces cerevisiae wild-type strain 2805 and L3262, andSaccharomyces cerevisiae mutants S28Y4, SLH15 (yps3Δ), SLH16(yps1Δ/yps3Δ), SLH17 (yps2Δ/yps3Δ), and SLH18 (yps1Δ/yps2Δ/yps3Δ) weretransformed with the expression vector pG10-hPTH1, followed by theselection of Ura⁺ transformants. These recombinant yeast strains capableof hPTH expression were subjected to hPTH expression analysis.

The yeast mutants were pre-cultured at 30° C. for 24 hours in minimalselective broths (amino acid-deficient yeast nitrogen substrate 0.67%,glucose 2%, casamino acid 0.5%). Each of the cultures was inoculated inan amount of 2% to a YPDG medium (yeast extract 1%, bacto peptone 2%,glucose 1%, galactose 1%) and then incubated at 30° C. for 48 hours.During this incubation, samples were withdrawn at 24 hours and 48 hours.500 μl of each sample was centrifuged at 5,000 rpm for 5 min to separatea supernatant from biomass. DOC(deoxycholic acid) and TCA(trichloroacetic acid) each was added in the amount of one-tenth volumeof the supernatant, which was then allowed to stand at 0° C. for 30 minto precipitate the proteins contained therein. Centrifugation dt 12,000rpm for 10 min produced a pellet which was then washed with acetone toremove the TCA solution remaining in the precipitated protein, anddissolved in 25 μl of a lysis buffer, followed by heating at 100° C. for5 min to obtain an extracellular protein fraction. 10 μl of theextracellular protein fraction was loaded on a 15% polyacrylamideseparating gel (pH 8.8, 10 cm wide, 8 cm length, 0.7 mm thick) which wasthen subjected to electrophoresis at 125 V and 25 mA for 1.5 hours. Theproteins run on the gel were visualized by Coumassie blue. Theelectrophoresis results are shown for the 24-hour sample in FIG. 3 a andfor the 48-hours sample in FIG. 3 b.

As seen in these electrophoresis photographs, the recombinant hPTHsecreted from the Saccharomyces cerevisiae strains are separated onSDS-PAGE, appearing as three bands: i for the intact molecule (1-84);.d1 for an N-terminus truncated molecule (27-84); and d2 for a C-terminustruncated molecule (1-80). The proteins located in the i band and d1band were transferred onto a PVDF (polyvinylidene difluoride) membrane.N-terminal amino acid sequencing of the proteins with the aid ofMilligenlBiosearch M 600 protein sequencer identified the amino acidsequences Ser-Val-Ser-Glu-Ile (SEO ID NO: 5) and Lys-Leu-Gln-Asp-Val(SEQ ID NO: 6) at the N-terminal regions of the proteins of the i and d1bands, respectively, indicating that the protein of the i band is theintact molecule of hPTH while the protein of the d1 band is a truncatedform (27-84) which is lacking in 26 amino acid residues of theN-terminal region of hPTH. As for the d2 band, its protein, thoughshowing an electrophoretic band of 14 kDa larger than the intactmolecule, was found to be a truncated hPTH form lacking 4 to 5 aminoacid residues of the C-terminus (1-79, 80) as analyzed by HPLC, MALDImass spectrometry, and C-terminal amino acid sequencing. Thecharacteristic electrophoretic pattern is reported to be attributed tothe conformational change of the protein resulting from the removal ofthe C terminus (Vad et al., Protein Expr. Purif 13:396-402 (1998)).

EXAMPLE 3 hPTH Expression Patterns in Yapsin Protease-Deficient Mutants

To examine the effect of the disruption of YPS1 and YPS3 genes on hPTHexpression, hPTH molecules secreted from the non-transformed, wild-typeSaccharomyces cerevisiae 2805, the transformed wild-type Saccharomyces2805/pG10-hPTH1, the transformed yps1Δ mutant S28Y3/pG10-hPTH1, and thetransformed yps3Δ mutant S28Y4/pG10-hPTH1 were subjected to SDS-PAGEelectrophoresis. The electrophoresis results are shown in FIG. 3.

As seen in the electrophoresis photographs, no hPTH-molecules wereexpressed in the non-transformed wild-type strain (lane 1). About 50% ormore of the hPTH expressed in the transformed wild-type strain 2805existed in the truncated d1 form (lanes 3 and 4). In the case of theyps1Δ mutant S28Y3, the truncated forms of the hPTH secreted into themedia comprised 5% or less of the total hPTH when it was obtained fromthe 24-hour culture. In contrast, as much as about 30-40% of the hPTHobtained from the 48-hour culture was found to exist in the d1 form. Thestrain S28Y4, which was disrupted at YPS3 gene only, was not differentfrom the wild type strain in hPTH expression patterns: about 50% or moreof the hPTH secreted within 24 hours was found to exist in truncatedforms.

The results, taken together and shown in FIG. 3 demonstrate that themain protease causative of the cleavage of hPTH in the early culturestage is Yapsin 1 and other proteases than Yapsin 1 are responsible forthe cleavage of hPTH in the late culture stage. However, In the case ofthe strain that is disrupted at the YPS3 gene only, no significanteffects can be obtained because the activity of Yapsin 1 already cleavesthe hPTH to a significant extent in the early culture stage.

Accordingly, an examination was made of the expression pattern of hPTHproduction when the YPS3 gene was disrupted in combination with otherYapsin genes YPS1 and YPS2. To this end, hPTH was expressed in thenon-transformed wild-type Saccharomyces cerevisiae L3262 serving as acontrol, the transformed L3262/pG10-hPTH1, the transformed yps3Δ mutantSLH15/pG10-hPTH1, the transformed yps1Δ/yps3Δ mutant SLH16/pG10-hPTH1,the transformed yps2Δ/yps3Δ mutant SLH17/pG10-hPTH1, and transformedyps1Δ/yps2Δ/yps2Δ mutant SLH18/pG10-hPTH1. Secreted proteins wereelectrophoresed in the same manner as above and the electrophoresisresults are shown for the sample withdrawn after 24 hours of incubationin FIG. 4 a and for the sample withdrawn after 48 hours of incubation inFIG. 4 b.

As seen in FIG. 4, no hPTH was detected in the non-transformed strain(lane 1). As much as 50% of the hPTH secreted from the transformedwild-type strain (lanes 2 and 3), YPS3-disrupted strain (lanes 4 and 5),and YPS2 and YPS3-double disrupted strain (lane 6 and 7) existed in theN-terminus truncated d1 form when collected at 24 hours. After 48 hours,hPTH cleavage was found to be further aggravated to increase theproportion of truncated hPTH. On the other hand, interestingly, none ofthe hPTH molecules secreted from the YPS1 and YPS3-double disruptedstrain (lanes 6 and 7) and YPS1, PYS2, YPS3-triple disrupted strain(lanes 10 and 11) had the N-terminus truncated d1 form even if they wereobtained at 48 hours. In addition, it was revealed that the truncatedform of d2 band comprised about 20% of the total hPTH secreted from theYPS1 and YPS3-double disrupted strain, while intact hPTH of the i band,but no d2 band protein, was observed even after 48 hours culture of theYPS1/YPS2/YPS3-triple disrupted strain. These results, taken together,showed that the hPTH degradation problem that the wild-type yeaststrains have can be almost completely solved by use of the strain inwhich all of YPS1, YPS2 and YPS3 genes are disrupted.

EXAMPLE 4 hPTH Expression Patterns in High-Concentration Culture

An experiment was carried out to examine whether the hPTH expressed fromthe yapsin protease-deficient mutants created as above underwentproteolysis or not. In this regard, galactose was continually fed intothe flask to imitate the conditions for high-concentration cultures in afermentation bath. After pre-incubation for 24 hours in YPDG broths, thestrains were further cultured for 72 hours while galactose was fed tothe broths at intervals of 24 hours to maintain the galactoseconcentration of the broths at the level of 2%.

Under these conditions, hPTH was expressed in the non-transformedwild-type Saccharomyces cerevisiae L3262 serving as a control, thetransformed L3262/pG10-hPTH1, the transformed yps1Δ mutantSLH11/pG10-hPTH1, the transformed yps1Δ/yps3Δ mutant SLH16/pG10-hPTH1,and transformed yps1Δ/yps2Δ/yps2Δ mutant SLH18/pG10-hPTH1. Secretedproteins were electrophoresed in the same manner as above and theelectrophoresis results are shown for the strains L3262/pG10-hPTH1 andSLH11/pG10-hPTH1 in FIG. 5 a and for the strains SLH16/pG10-hPTH1 andSLH18/pG10-hPTH1 in FIG. 5 b.

As seen in FIG. 5, as much as 50% of the hPTH secreted from the wildtype yeast (lanes 2-7, FIG. 5 a) already existed in the N-terminustruncated d1 form when collected at 24 hours. On the other hand, in thecase of the cultures of the yps1Δ mutant (FIG. 5 a, lanes 8-13) and theyps1Δ/yps3Δ mutant (lanes 2-7, FIG. 5 b), hPTH was not degraded in the24 hour cultures, while hPTH degradation was observed to be significantin the 48 hour high-concentration culture to which galactose had beenprovided continually. In contrast, the yps1Δyps2Δyps3Δ mutant cultures(lanes 8-13, FIG. 5 b) were found to have no hPTH of the N-terminustruncated d1 form with significant increase of intact hPTH (i band) evenwhen collected at 72 hours. Still even at 144 hours, the cultures of thetriple-disrupted mutant were observed to have no truncated forms ofhPTH. These results clearly demonstrated that the hPTH degradationproblem which occurs in the late high-concentration culture stage can becompletely solved by employing the mutant in which all of YPS1, YPS2 andYPS3 genes are disrupted.

As described hereinbefore, yeast mutants which are disrupted both inYapsin 1 (previously YPA3) and Yapsin 3 genes (yps1Δ/yps3Δ) or all inYapsin 1, Yapsin 2 (previously MKC7) and Yapsin3 genes(yps1Δ/yps2Δ/yps3Δ) can secrete intact hPTH, useful as therapeutics forvarious disorders, at high yield by virtue of their inability to degradethe hormonal peptide.

The present invention has been described in an illustrative manner, andit is to be understood that the terminology used is intended to be inthe nature of description rather than of limitation. Many modificationsand variations of the present invention are possible in light of theabove teachings. Therefore, it is to be understood that within the scopeof the appended claims, the invention may be practiced otherwise than asspecifically described.

1. A method for producing a recombinant human parathyroid hormone (hPTH)from a Saccharomyces cerevisiae host, comprising the steps of:subjecting the Saccharomyces cerevisiae host to a deletion mutation inthe genes encoding Yapsin 1 (YPS1) and Yapsin 3 (YPS3); transforming theSaccharomyces cerevisiae host with an expression vector containing agene encoding human parathyroid hormone; and culturing the transformedSaccharomyces cerevisiae host in an appropriate medium.
 2. The method asset forth in claim 1, wherein the deletion mutation is carried out bydisrupting the genes encoding Yapsin 1 and Yapsin 3 by use of aSaccharomyces cerevisiae selection marker.
 3. The method as set forth inclaim 1, wherein the Saccharomyces cerevisiae host is further disruptedin the Yapsin 2 (YPS2) gene.
 4. A Saccharomyces cerevisiae strainprepared by transforming a Saccharomyces cerevisiae yps1Δyps3Δmutantwith an expression vector anchoring a human parathyroid hormone gene. 5.The Saccharomyces cerevisiae strain as set forth in claim 4, wherein theSaccharomyces cerevzsiae strain is Saccharomyces cerevisiaeSLH16/pG10-hPTH (accession No. KCTC 0815BP) and the expression vector ispG10-hPTH1.
 6. A Saccharomyces cerevisiae strain, prepared bytransforming a Saccharomyces cerevisiae yps1Δyps2Δyps3A mutant with anexpression vector anchoring a human parathyroid hormone gene.
 7. TheSaccharomyces cerevisiae strain as set forth in claim 6, wherein theSaccharomyces cerevisiae strain is Saccharomyces cerevisiaeSLH18/pG10-hPTH (accession No. KCTC 0816BP) and the expression vector ispG10-hPTH1.